PROJECT SUMMARY/ABSTRACT Staphylococcus aureus is responsible for a large number of hospital- and community-acquired infections worldwide. Therapeutic options to combat S. aureus infections are limited due to the high level of antibiotic resistance and lack of an effective vaccine. Thus, there is a significant need for the development of effective therapeutics against this organism. Critical to the pathogenic lifestyle of S. aureus is the killing of phagocytes; innate immune cells integral to the control of Staphylococcal infections. Thus, the long-term goal of this research program is to understand the mechanism employed by S. aureus to injure these critical immune cells. We have described that the leukocidin A/B (LukAB) plays an essential role in protecting S. aureus from phagocyte-mediated killing by targeting and eliminating these cells. Our work has established that LukAB is present in all clinical strains, yet is the most divergent member of the bi-component pore-forming family of toxins; is responsible for S. aureus-mediated demise of primary human phagocytes during ex vivo infections; targets the CD11b integrin; exhibits species specificity by preferentially targeting the human I-domain of CD11b over the murine I-domain; is produced in vivo during both murine and human infections; and kills human leukocytes when produced by S. aureus from both extracellular and intracellular environments. The primary goals of this application are to: elucidate the mechanism by which LukAB binds to human CD11b in order to define the species specificity determinants to develop an improved murine model for S. aureus infection (Aim 1); define the mechanism by which LukAB kills human phagocytes (Aim 2); and delineate the contribution of LukAB variants to the pathogenesis of other S. aureus clones (Aim 3). To accomplish these Aims, we propose to employ a multidisciplinary approach that combines molecular biology, genetics, cellular immunology, whole genome screens, and biochemistry, together with ex vivo and in vivo infection models. Understanding the molecular details of how LukAB mediates targeting and killing of phagocytes will provide insight into how S. aureus bi-component pore-forming toxins kill host cells and the importance of these toxins to S. aureus pathogenesis.